INTRACELLULAR PH REGULATION IN ISCHEMIC MYOCARDIUM

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DESCRIPTION (adapted from the applicant's abstract): By combining results from 31P NMR spectroscopy of the rat heart and conventional analysis of freeze-clamped tissue the applicant has obtained evidence for a regulatory role for intracellular pH (pHi) on adenine nucleotide resynthesis in myocardium improved by ischemia. Specifically, by combining high-flow hypoxia (where pHi does not fall below 7.0) and ischemia (where pH falls to 6), the applicant found that low pHi leads to greater accumulation of AMP during ischemia and to greater ATP resynthesis and improved functional recovery during reflow. Following 28 minutes of global normothermic ischemia, the amount of ATP synthesized upon reperfusion equals the amount of AMP that had accumulated during ischemia. Essentially all of the ATP resynthesized during reperfusion is via this mechanism. The applicant hypothesizes that the underlying mechanism for this observation is inhibition of cytoplasmic AMP-specific 5'-nucleotidase (5'-NT) by H+, thereby, preventing the conversion of AMP to adenosine. If this is the case, low pHi protects the myocardium by preserving a significant fraction of the cytosolic adenine nucleotide pool during ischemia. Using NMR spectroscopy of isolated perfused rat heart and conventional biochemical tools, the applicant will pursue three specific aims which focus on the consequences of acidic pH on the capacity of the ischemic myocardium to synthesize ATP during reperfusion: (1) to define in vivo regulation of 5'-NT activity; (2) to determine the time course of activation, inhibition and reactivation of 5'-NT activity; (3) to define the relationship among pHi, ATP and return of cardiac function.